These studies are designed to study the activation of human neutrophils by phagocytic and non-phagocytic stimuli, with particular reference to the role of membrane lipids as transducers or modulators of stimulus-response coupling. The time course of changes in endogenous lipids during the activation of human neutrophils will be determined and correlated with changes in CA++ translocation, cAMP response, prostanoid generation, aggregation, degranulation and superoxide anion generation. We will identify which phosphatide levels are changed and how the time course of these changes correlates with the "onset" and "cessation" of aggregation, superoxide anion generation and degranulation. We will assess if the lipd changes are a consequence of receptor ligand interaction or if they can be initiated by CA++ ionophores such as A23187 or ionomycin. The role of the phosphatidyl inositol/phosphatidic acid (PI/PA) cycle in CA++ translocation during neutrophil activation will be critically evaluated. The possibility will be examined that lipid derivatives act to translocate CA++ by serving as "endogenous CA++ ionophores." The alternative hypothesis will also be considered that lipid changes modulate the activity of a protein "CA++ gate." The source of arachidonic acid and therefore of prostanoids during neutrophil activation will be determined. The role of arachidonate metabolites as mediators and modulators of the activation sequence will be evaluated, in particular the hypothesis that a lipoxygenase product is concerned in CA++ translocation. A sensitive assay system (devised in our laboratory) employing liposome-entrapped arsenazo III will be used to monitor ionophore activity of lipid intermediates, to compare the rate constants and stoichiometry of the transport process in artificial systems with that of the human neutrophil. Since the appropriate functioning of the neutrophil is important in host defenses, as well as in inflammatory disorders, a more fundamental understanding of the activation sequence of the neutrophil is of obvious clinical importance.